Ribosome profiling is a method based on deep sequencing of ribosome-protected mRNA fragments. Ribosome profiling, also known as Ribo-Seq (ribosome sequencing) or ART-Seq (active mRNA translation sequencing), provides a “snapshot” of all the ribosomes active in a cell at a specific time point. This information can help researchers determine which proteins are being actively translated in a cell.
Ribosome profiling analysis enables systematic monitoring of cellular translation processes and prediction of protein abundance. Determining what regions of a message are being translated can help define the proteome of complex organisms. In combination with massively parallel high-throughput next-generation sequencing (NGS), ribosome profiling with Ribo-Seq allows detailed and accurate in vivo analysis of protein production.
By identifying transcripts undergoing active translation, Ribo-Seq can provide visibility into an important aspect of gene regulation.
Ribosome profiling protocols often use size-exclusion chromatography. This method streamlines the steps to convert ribosome-protected RNA fragments into libraries compatible with sequencing on Illumina instruments. It is a simple, rapid, and scalable method that does not require special equipment. In contrast, other methods to isolate polysomes and monosomes rely on several hours of ultracentrifugation using either a sucrose gradient or a cushion.
Various ribosome profiling publications featuring Illumina sequencing instruments are available as a reference. For example, the following Nature Cell Biology publication discusses the use of Ribo-Seq to study early neurodevelopment. The paper includes a Ribo-Seq sample preparation and data analysis protocol and indicates that sequencing was performed on an Illumina NovaSeq 6000 System. In addition, Illumina no longer sells Ribo-Seq library preparation kits, but Ribo-Seq kits optimized for Illumina sequencing are available from other companies such as Immagina Biotech.
Read publication: Ribosome levels and neurodevelopment
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Ribosome profiling reagent details
Illumina TruSeq Ribo Profile kits (discontinued) were based on the protocol and reagents described in this Nature Protocols paper.
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